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Key points Mitochondrial Ca2+ uptake stimulates the Krebs cycle to regenerate the reduced forms of pyridine nucleotides (NADH, NADPH and FADH2 ) required for ATP production and reactive oxygen species (ROS) elimination. Ca2+ /calmodulin-dependent protein kinase II (CaMKII) has been proposed to regulate mitochondrial Ca2+ uptake via mitochondrial Ca2+ uniporter phosphorylation. We used two mouse models with either global deletion of CaMKIIδ (CaMKIIδ knockout) or cardiomyocyte-specific deletion of CaMKIIδ and γ (CaMKIIδ/γ double knockout) to interrogate whether CaMKII controls mitochondrial Ca2+ uptake in isolated mitochondria and during β-adrenergic stimulation in cardiac myocytes. CaMKIIδ/γ did not control Ca2+ uptake, respiration or ROS emission in isolated cardiac mitochondria, nor in isolated cardiac myocytes, during β-adrenergic stimulation and pacing. The results of the present study do not support a relevant role of CaMKII for mitochondrial Ca2+ uptake in cardiac myocytes under physiological conditions. Abstract Mitochondria are the main source of ATP and reactive oxygen species (ROS) in cardiac myocytes. Furthermore, activation of the mitochondrial permeability transition pore (mPTP) induces programmed cell death. These processes are essentially controlled by Ca2+ , which is taken up into mitochondria via the mitochondrial Ca2+ uniporter (MCU). It was recently proposed that Ca2+ /calmodulin-dependent protein kinase II (CaMKII) regulates Ca2+ uptake by interacting with the MCU, thereby affecting mPTP activation and programmed cell death. In the present study, we investigated the role of CaMKII under physiological conditions in which mitochondrial Ca2+ uptake matches energy supply to the demand of cardiac myocytes. Accordingly, we measured mitochondrial Ca2+ uptake in isolated mitochondria and cardiac myocytes harvested from cardiomyocyte-specific CaMKII δ and γ double knockout (KO) (CaMKIIδ/γ DKO) and global CaMKIIδ KO mice. To simulate a physiological workload increase, cardiac myocytes were subjected to β-adrenergic stimulation (by isoproterenol superfusion) and an increase in stimulation frequency (from 0.5 to 5 Hz). No differences in mitochondrial Ca2+ accumulation were detected in isolated mitochondria or cardiac myocytes from both CaMKII KO models compared to wild-type littermates. Mitochondrial redox state and ROS production were unchanged in CaMKIIδ/γ DKO, whereas we observed a mild oxidation of mitochondrial redox state and an increase in H2 O2 emission from CaMKIIδ KO cardiac myocytes exposed to an increase in workload. In conclusion, the results obtained in the present study do not support the regulation of mitochondrial Ca2+ uptake via the MCU or mPTP activation by CaMKII in cardiac myocytes under physiological conditions.

Funder: Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein‐Westfalen; Id: http://dx.doi.org/10.13039/501100009591, Funder: Bundesministerium für Bildung und Forschung; Id: http://dx.doi.org/10.13039/501100002347, AIMS: MICU1 encodes the gatekeeper of the mitochondrial Ca2+ uniporter, MICU1 and biallelic loss-of-function mutations cause a complex, neuromuscular disorder in children. Although the role of the protein is well understood, the precise molecular pathophysiology leading to this neuropaediatric phenotype has not been fully elucidated. Here we aimed to obtain novel insights into MICU1 pathophysiology. METHODS: Molecular genetic studies along with proteomic profiling, electron-, light- and Coherent anti-Stokes Raman scattering microscopy and immuno-based studies of protein abundances and Ca2+ transport studies were employed to examine the pathophysiology of MICU1 deficiency in humans. RESULTS: We describe two patients carrying MICU1 mutations, two nonsense (c.52C>T; p.(Arg18*) and c.553C>T; p.(Arg185*)) and an intragenic exon 2-deletion presenting with ataxia, developmental delay and early onset myopathy, clinodactyly, attention deficits, insomnia and impaired cognitive pain perception. Muscle biopsies revealed signs of dystrophy and neurogenic atrophy, severe mitochondrial perturbations, altered Golgi structure, vacuoles and altered lipid homeostasis. Comparative mitochondrial Ca2+ transport and proteomic studies on lymphoblastoid cells revealed that the [Ca2+ ] threshold and the cooperative activation of mitochondrial Ca2+ uptake were lost in MICU1-deficient cells and that 39 proteins were altered in abundance. Several of those proteins are linked to mitochondrial dysfunction and/or perturbed Ca2+ homeostasis, also impacting on regular cytoskeleton (affecting Spectrin) and Golgi architecture, as well as cellular survival mechanisms. CONCLUSIONS: Our findings (i) link dysregulation of mitochondrial Ca2+ uptake with muscle pathology (including perturbed lipid homeostasis and ER-Golgi morphology), (ii) support the concept of a functional interplay of ER-Golgi and mitochondria in lipid homeostasis and (iii) reveal the vulnerability of the cellular proteome as part of the MICU1-related pathophysiology.

Due to its Ca(2+) buffering capacity, the mitochondrion is one of the most important intracellular organelles in regulating Ca(2+) dynamic oscillation. Mitochondrial calcium uniporter (MCU) is the primary mediator of Ca(2+) influx into mitochondria, manipulating cell energy metabolism, ROS production, and programmed cell death, all of which are critical for carcinogenesis. The understanding of the uniporter complex was significantly boosted by recent groundbreaking discoveries that identified the uniporter pore‐forming subunit MCU and its regulatory molecules, including MCU‐dominant negative β subunit (MCUb), essential MCU regulator (EMRE), MCU regulator 1 (MCUR1), mitochondrial calcium uptake (MICU) 1, MICU2, and MICU3. These provide the means and molecular platform to investigate MCU complex (uniplex)‐mediated impaired Ca(2+) signalling in physiology and pathology. This review aims to summarize the progress of the understanding regulatory mechanisms of uniplex, roles of uniplex‐mediated Ca(2+) signalling in cancer, and potential pharmacological inhibitors of MCU.

The polymer of intrinsic microporosity PIM-EA-TB provides a molecularly rigid micropore structure containing tertiary amine sites and is shown here to interact with hydrogen bonding guest molecules such as caffeic acid. Voltammetric data with a PIM-EA-TB film on glassy carbon electrodes show that in both acidic solution (pH 2; PIM-EA-TB is protonated) and in neutral solution (pH 6; PIM-EA-TB is not protonated) caffeic acid is slowly accumulated into the microporous host. Binding constants are estimated and suggested to be linked to hydrogen bonding causing accumulation of caffeic acid. When employing PIM-EA-TB as an asymmetric membrane coated onto a 5 μm thick Teflon support film with 10 μm diameter microholes (using either a single microhole or a 10×10 array of microholes), binding of caffeic acid is shown to cause a modulation of the ionic current without affecting the pH-dependent ionic diode behaviour. Two complementary types of effects of caffeic acid guests are discussed based on blocking anion diffusion pathways and based on removal of positive charges. The caffeic acid transport mechanism/efficiency is investigated in view of selective molecular pumping.

BACKGROUND AND PURPOSE Treatment of cardiac arrhythmia remains challenging due to severe side effects of common anti-arrhythmic drugs. We previously demonstrated that mitochondrial Ca2+ uptake in cardiomyocytes represents a promising new candidate structure for safer drug therapy. However, druggable agonists of mitochondrial Ca2+ uptake suitable for preclinical and clinical studies are still missing. EXPERIMENTAL APPROACH Herewe screened 727 compounds with a history of use in human clinical trials in a three-step screening approach. As a primary screening platform we used a permeabilized HeLa cell-based mitochondrial Ca2+ uptake assay. Hits were validated in cultured HL-1 cardiomyocytes and finally tested for anti-arrhythmic efficacy in three translational models: a Ca2+ overload zebrafish model and cardiomyocytes of both a mouse model for catecholaminergic polymorphic ventricular tachycardia (CPVT) and induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. KEY RESULTS We identifiedtwo candidate compounds, the clinically approved drugs ezetimibe and disulfiram, which stimulate SR-mitochondria Ca2+ transfer at nanomolar concentrations. This is significantly lower compared to the previously described mitochondrial Ca2+ uptake enhancers (MiCUps) efsevin, a gating modifier of the voltage-dependent anion channel 2, and kaempferol, an agonist of the mitochondrial Ca2+ uniporter. Both substances restored rhythmic cardiac contractions in a zebrafish cardiac arrhythmia model and significantly suppressed arrhythmogenesis in freshly isolated ventricular cardiomyocytes from a CPVT mouse model as well as induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. CONCLUSION AND IMPLICATIONS Taken together we identified ezetimibe and disulfiram as novel MiCUps and efficient suppressors of arrhythmogenesis and as such as, promising candidates for future preclinical and clinical studies.

Opioid-induced hyperalgesia (OIH) and allodynia is a well-known phenomenon and refers to the pain sensitization in patients after prolonged opioid exposure. OIH limits the use of opioids in pain control, but the underlying mechanisms are not fully clear. This study investigated the role of mitochondrial Ca2+ uniporter (MCU) in remifentanil (a commonly used opioid analgesic)-induced allodynia. Using a rat model of OIH, we found that incision- and remifentanil-induced mechanical allodynia were remarkably attenuated by pretreatment with Ru360, a specific MCU antagonist, suggesting a critical role of MCU in both incision- and opioid-induced allodynia. In addition, imaging studies with Rhod-2 (a mitochondrial Ca2+ dye) in spinal tissues demonstrated increased mitochondrial Ca2+ level in response to incision and remifentanil infusion, which was attenuated by Ru360. Western blot and immunohistochemistry showed that pNR [phosphorylated N-methyl-D-aspartate (NMDA) receptor] and pERK (phosphorylated extracellular signal-regulated kinase) are increased during both incision-induced hyperalgesia and remifentanil-induced hyperalgesia, and again the increases in pNR and pERK were remarkably attenuated by Ru360. Together, our data demonstrate that MCU plays a critical role in remifentanil-induced postoperative mechanical allodynia, with NMDA receptor and ERK as possible downstream effectors. Our findings provide novel mechanisms for remifentanil-induced mechanical allodynia and encourage future studies to examine the mitochondrial Ca2+ uniporter as a potential therapeutic target for prevention of OIH.

Mitochondria play a significant role in shaping cytosolic Ca2+ signals. Thus, transfer of Ca2+ across the mitochondrial membrane is much studied, not only in intact cells but also in artificial systems such as mitochondrial suspensions or permeabilised cells. Observed rates of Ca2+ changes vary by at least one order of magnitude. In this work, we investigate the relationship between the Ca2+ dynamics observed in various experimental conditions using a computational model calibrated on experimental data. Results confirm that mitochondrial Ca2+ exchange fluxes through the mitochondrial Ca2+ uniporter (MCU) and the Na+ /Ca2+ exchanger obey the same basic kinetics in cells and in suspensions, and emphasise the important role played by the high Ca2+ levels reached in mitochondria-associated endoplasmic reticulum membranes in intact cells. Tissue specificity can be ascribed to the different modes of regulation of the MCU by Ca2+ , probably related to the specific levels of expression of the Ca2+ sensing regulator subunit of this channel. The model emphasises the importance of mitochondrial density and buffering in controlling the rate of Ca2+ exchanges with mitochondria, as verified experimentally. Finally, we show that heterogeneity between individual mitochondria can explain the large range of amplitudes and rates of rise in mitochondrial Ca2+ concentration that have been observed experimentally.

Podocytes are component cells of the glomerular filtration barrier, and their loss by apoptosis is the main cause of proteinuria that leads to diabetic nephropathy (DN). Therefore, insights into podocyte apoptosis mechanism would allow a better understanding of DN pathogenesis and thus help develop adequate therapeutic strategies. Here, we investigated the molecular mechanism of palmitic acid-inhibited cell death in mouse podocytes, and found that palmitic acid increased cell death in a dose- and time-dependent manner. Palmitic acid induces apoptosis in podocytes through upregulation of cytosolic and mitochondrial Ca2+, mitochondrial membrane potential (MMP), cytochrome c release, and depletion of endoplasmic reticulum (ER) Ca2+. The intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N, N′-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), partially prevented this upregulation whereas 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor; dantrolene, a ryanodine receptor (RyR) inhibitor; and 4,4′-diisothiocyanatostibene-2,2′-disulfonic acid (DIDS), an anion exchange inhibitor, had no effect. Interestingly, ruthenium red and Ru360, both inhibitors of the mitochondrial Ca2+ uniporter (MCU), blocked palmitic acid-induced mitochondrial Ca2+ elevation, cytochrome c release from mitochondria to cytosol, and apoptosis. siRNA to MCU markedly reduced palmitic acid-induced apoptosis. These data indicate that Ca2+ uptake via mitochondrial uniporter contributes to palmitic acid-induced apoptosis in mouse podocytes. J. Cell. Biochem. 118: 2809–2818, 2017. © 2017 Wiley Periodicals, Inc.

Ca2+ and mitochondria are inextricably linked to cardiac function and dysfunction. Ca2+ is central to cardiac excitation-contraction coupling and stimulates mitochondrial energy production to fuel contraction. Under pathological conditions of dysregulated Ca2+ cycling, mitochondrial Ca2+ overload activates cellular death pathways. Thus, in the cardiomyocyte, the mitochondrial Ca2+ microdomain is where contraction, energy and death collide. A key component of mitochondrial Ca2+ signalling is the mitochondrial Ca2+ uniporter complex (uniplex), an inner membrane Ca2+ transporter and major pathway of mitochondrial Ca2+ entry. Once known only as the unidentified target for ruthenium red and related compounds, in recent years, the uniplex has evolved into a complex multiprotein assembly. The identification of the molecular constituents of the uniplex has made possible the generation of targeted genetic models to interrogate uniplex function in vivo. This review will summarize our current understanding of the molecular structure of the uniplex, its impact on mitochondrial energetics and cardiac physiology, its contribution to cardiomyocyte death, and its expanding roles in cardiac biology.

Uniporters are a large class of transporters mediating facilitated diffusion of substrates along the direction of the substrate concentration gradient. Recently, structures of several important uniporters have been reported; however, the precise mechanisms of uniporter function remain subject of debate. Here, we present a series of general thermodynamic descriptions of uniporters, aimed at understanding the structure-function relationship of uniporters, and in particular to reconcile biochemical phenomena of uniporters with our previously proposed thermodynamic model of general transporters.

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca(2+) in hesperidin-induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca(2+) level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol-1,4,5-triphosphate receptor (IP3 R) and ryanodine receptor also block the mitochondrial Ca(2+) accumulation suggesting that the release of Ca(2+) from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca(2+) level. Pretreatment with ruthenium red (RuRed), a Ca(2+) uniporter inhibitor inhibited the hesperidin-induced mitochondrial Ca(2+) overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca(2+) influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3 R and ryanodine receptor mediated release of Ca(2+) from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin-induced paraptosis in HepG2 cells.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by pulmonary vascular obstruction due in part to excessive pulmonary artery endothelial cells (PAECs) migration and proliferation. The mitochondrial fission protein dynamin-related protein-1 (DRP1) has important influence on pulmonary vascular remodeling. However, whether DRP1 participates in the development and progression of pulmonary vascular angiogenesis has not been reported previously. To test the hypothesis that DRP1 promotes the angiogenesis via promoting the proliferation, stimulating migration, and inhibiting the apoptosis of PAECs in mitochondrial Ca(2+)-dependent manner, we performed following studies. Using hemodynamic analysis and morphometric assay, we found that DRP1 mediated the elevation of right ventricular systemic pressure (RVSP), right heart hypertrophy, and increase of pulmonary microvessels induced by hypoxia. DRP1 inhibition reversed tube network formation in vitro stimulated by hypoxia. The mitochondrial Ca(2+) inhibited by hypoxia was recovered by DRP1 silencing. Moreover, pulmonary vascular angiogenesis promoted by DRP1 was reversed by the specific mitochondrial Ca(2+) uniporter inhibitor Ru360. In addition, DRP1 promoted the proliferation and migration of PAECs in mitochondrial Ca(2+)-dependent manner. Besides, DRP1 decreased mitochondrial membrane potential, reduced the DNA fragmentation, and inhibited the caspase-3 activation, which were all aggravated by Ru360. Therefore, these results indicate that the mitochondrial fission machinery promotes migration, facilitates proliferation, and prevents from apoptosis via mitochondrial Ca(2+)-dependent pathway in endothelial cells leading to pulmonary angiogenesis.

New Findings What is the central question of this study? Hyperthyroidism is a cardiac risk factor, but thyroid therapy is used on myocardial stunning. What is the consequence of hyperthyroidism for mitochondrial metabolism and Ca2+ handling of the postischaemic stunned heart? What is the main finding and its importance? Hyperthyroidism reduced stunning and improved muscle economy of the postischaemic rat heart. The activities of the mitochondrial sodium–calcium exchanger and mitochondrial K+ channel in hyperthyroid rat hearts were different from those in the euthyroid rat hearts. These findings contribute to the understanding of mitochondrial bioenergetics in pathology and support thyroid therapy in the stunning induced by ischaemia. Transient ischaemia and hyperthyroidism are cardiovascular risk factors. Nevertheless, 3,5,3′-triiodothyronine/thyroxine therapy has been used to revert myocardial stunning. We studied the influence of hyperthyroidism on the role played by mitochondria in myocardial stunning consequent to ischaemia–reperfusion. Rats were injected s.c. daily with 20 μg kg−1 triiodothyronine for 15 days (HpT group). Isolated ventricles from either HpT or euthyroid (EuT) rats were perfused in a calorimeter, and left intraventricular pressure (in millimetres of mercury) and heat release (Ht; in milliwatts per gram) were measured. Stunning was evoked by 20 min of no-flow ischaemia and 45 min reperfusion. The HpT hearts developed higher postischaemic contractile recovery (PICR) and improved total muscle economy (P/Ht) with lower diastolic contracture (ΔLVEDP) than EuT hearts. Release of Ca2+ from the sarcoplasmic reticulum during reperfusion with 10 mm caffeine in low-[Na+] Krebs solution evoked a higher contracture in EuT than in HpT hearts. Blockade of the mitochondrial sodium–calcium exchanger with clonazepam increased ΔLVEDP and reduced P/Ht and PICR in HpT but not in EuT hearts. The clonazepam-induced dysfunction in HpT hearts was reduced by ciclosporin, suggesting a dependance on activation of the mitochondrial permeability transition pore. Blockade of the mitochondrial Ca2+ uniporter with Ru360 reduced P/Ht and PICR to ∼10% in both HpT and EuT hearts. Blockade of mitochondrial K+ channels with 5-hydroxydecanoate increased LVEDP and reduced PICR and P/Ht in HpT hearts, while it only increased LVEDP in EuT hearts. The results suggest that hyperthyroidism prevents the stunning with high dependence on the mitochondrial sodium–calcium exchanger and mitochondrial K+ channels. Both HpT and EuT hearts showed a similar and critical role of the uniporter. The HpT hearts have a slow sarcoplasmic reticulum Ca2+ loss and low mitochondrial Ca2+ uptake.

Key points Lat4 (Slc43a2) transports branched-chain amino acids, phenylalanine and methionine, and is expressed in kidney tubule and small intestine epithelial cells. Using a new knockout model as a negative control, it is shown that Lat4 is expressed at the basolateral side of small intestine enterocytes and kidney epithelial cells of the proximal tubule, thick ascending limb and distal convoluted tubule. In the Xenopus oocyte expression system, Lat4 is shown to function as a uniporter with symmetric intracellular and extracellular apparent affinities for phenylalanine. Mice lacking Lat4 display a slight intrauterine growth restriction, postnatal malnutrition and early death, presumably as a result of defective amino acid (re)absorption. These results demonstrate the crucial role that the uniporter Lat4 plays for amino acid transport across cellular barriers and mouse development. Abstract Amino acid (AA) uniporter Lat4 (Slc43a2) mediates facilitated diffusion of branched-chain AAs, methionine and phenylalanine, although its physiological role and subcellular localization are not known. We report that Slc43a2 knockout mice were born at expected Mendelian frequency but displayed an ∼10% intrauterine growth retardation and low amniotic fluid AAs, suggesting defective transplacental transport. Postnatal growth was strongly reduced, with premature death occurring within 9 days such that further investigations were made within 3 days of birth. Lat4 immunofluorescence showed a strong basolateral signal in the small intestine, kidney proximal tubule and thick ascending limb epithelial cells of wild-type but not Slc43a2 null littermates and no signal in liver and skeletal muscle. Experiments using Xenopus laevis oocytes demonstrated that Lat4 functioned as a symmetrical low affinity uniporter with a K0.5 of ∼5 mm for both in- and efflux. Plasma AA concentration was decreased in Slc43a2 null pups, in particular that of non-essential AAs alanine, serine, histidine and proline. Together with an increased level of plasma long chain acylcarnitines and a strong alteration of liver gene expression, this indicates malnutrition. Attempts to rescue pups by decreasing the litter size or by nutrients injected i.p. did not succeed. Radioactively labelled leucine but not lysine given per os accumulated in the small intestine of Slc43a2null pups, suggesting the defective transcellular transport of Lat4 substrates. In summary, Lat4 is a symmetrical uniporter for neutral essential AAs localizing at the basolateral side of (re)absorbing epithelia and is necessary for early nutrition and development.

Dysregulation of mitochondrial Ca2+-dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca2+ transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1-mediated Ca2+ transport remains unresolved. This study examines LETM1-mediated mitochondrial Ca2+ transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf-Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca2+ influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ΔEF hand, D676A D688KLETM1 mutant-overexpressed cells, and in cells derived from patients with WHS. Although LETM1 levels were lower in WHS-derived fibroblasts, the mitochondrial Ca2+ uniporter components MCU, MCUR1, and MICU1 remain unaltered. In addition, the MCU mitoplast patch-clamp current (IMCU) was largely unaffected in LETM1-knockdown cells. Silencing of LETM1 also impaired basal mitochondrial oxygen consumption, possibly via complex IV inactivation and ATP production. Remarkably, LETM1 knockdown also resulted in increased reactive oxygen species production. Further, LETM1 silencing promoted AMPK activation, autophagy, and cell cycle arrest. Reconstitution of LETM1 or antioxidant overexpression rescued mitochondrial Ca2+ transport and bioenergetics. These findings reveal the role of LETM1-dependent mitochondrial Ca2+ flux in shaping cellular bioenergetics.—Doonan, P J., Chandramoorthy, H. C., Hoffman, N. E., Zhang, X., Cárdenas, C., Shanmughapriya, S., Rajan, S., Vallem, S., Chen, X., Foskett, J. K., Cheung, J. Y., Houser, S. R., Madesh, M. LETM1-dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation.

Background and Purpose Despite the importance of mitochondrial Ca2+ to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca2+ entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca2+ uniporter in an isolated heart model, at baseline and during increased workload following β-adrenoceptor stimulation. Experimental Approach Cardiac contractility, oxygen consumption and intracellular Ca2+ transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca2+ uniporter activity. Changes in mitochondrial Ca2+ content and energetic phosphate metabolite levels were determined. Key Results The addition of Ru360, a selective inhibitor of the mitochondrial Ca2+ uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 μM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360. Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca2+-dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca2+ content (2.5-fold). However, in Ru360-treated hearts, this parameter was attenuated. In addition, β-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca2+ handling, PKA or Ca2+/calmodulin-dependent PK signalling. Conclusions and Implications Inhibition of the mitochondrial Ca2+ uniporter decreases β-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca2+ uniporter activity.

Cardiac mitochondria can act as a significant Ca(2+) sink and shape cytosolic Ca(2+) signals affecting various cellular processes, such as energy metabolism and excitation-contraction coupling. However, different mitochondrial Ca(2+) uptake mechanisms are still not well understood. In this study, we analysed recently published Ca(2+) uptake experiments performed on isolated guinea pig cardiac mitochondria using a computer model of mitochondrial bioenergetics and cation handling. The model analyses of the data suggest that the majority of mitochondrial Ca(2+) uptake, at physiological levels of cytosolic Ca(2+) and Mg(2+), occurs through a fast Ca(2+) uptake pathway, which is neither the Ca(2+) uniporter nor the rapid mode of Ca(2+) uptake. This fast Ca(2+) uptake component was explained by including a biophysical model of the ryanodine receptor (RyR) in the computer model. However, the Mg(2+)-dependent enhancement of the RyR adaptation was not evident in this RyR-type channel, in contrast to that of cardiac sarcoplasmic reticulum RyR. The extended computer model is corroborated by simulating an independent experimental dataset, featuring mitochondrial Ca(2+) uptake, egress and sequestration. The model analyses of the two datasets validate the existence of two classes of Ca(2+) buffers that comprise the mitochondrial Ca(2+) sequestration system. The modelling study further indicates that the Ca(2+) buffers respond differentially depending on the source of Ca(2+) uptake. In particular, it suggests that the Class 1 Ca(2+) buffering capacity is auto-regulated by the rate at which Ca(2+) is taken up by mitochondria.

Communication between the plasma membrane and mitochondria is essential for initiating the Ca(2+) and metabolic signals required for secretion in β cells. Although voltage-dependent Na(+) channels are abundantly expressed in β cells and activated by glucose, their role in communicating with mitochondria is unresolved. Here, we combined fluorescent Na(+), Ca(2+), and ATP imaging, electrophysiological analysis with tetrodotoxin (TTX)-dependent block of the Na(+) channel, and molecular manipulation of mitochondrial Ca(2+) transporters to study the communication between Na(+) channels and mitochondria. We show that TTX inhibits glucose-dependent depolarization and blocks cytosolic Na(+) and Ca(2+) responses and their propagation into mitochondria. TTX-sensitive mitochondrial Ca(2+) influx was largely blocked by knockdown of the mitochondrial Ca(2+) uniporter (MCU) expression. Knockdown of the mitochondrial Na(+)/Ca(2+) exchanger (NCLX) and Na(+) dose response analysis demonstrated that NCLX mediates the mitochondrial Na(+) influx and is tuned to sense the TTX-sensitive cytosolic Na(+) responses. Finally, TTX blocked glucose-dependent mitochondrial Ca(2+) rise, mitochondrial metabolic activity, and ATP production. Our results show that communication of the Na(+) channels with mitochondria shape both global Ca(2+) and metabolism signals linked to insulin secretion in β cells.- Nita, I. I., Hershfinkel, M., Kantor, C., Rutter, G. A., Lewis, E. C., Sekler, I. Pancreatic β-cell Na(+) channels control global Ca(2+) signaling and oxidative metabolism by inducing Na(+) and Ca(2+) responses that are propagated into mitochondria.

Cross-talk between organelles and plasma membrane Ca(2+) channels is essential for modulation of the cytosolic Ca(2+) ([Ca(2+)]C) signals, but such modulation may differ among cells. In chromaffin cells Ca(2+) entry through voltage-operated channels induces calcium release from the endoplasmic reticulum (ER) that amplifies the signal. [Ca(2+)]C microdomains as high as 20-50 μm are sensed by subplasmalemmal mitochondria, which accumulate large amounts of Ca(2+) through the mitochondrial Ca(2+) uniporter (MCU). Mitochondria confine the high-Ca(2+) microdomains (HCMDs) to beneath the plasma membrane, where exocytosis of secretory vesicles happens. Cell core [Ca(2+)]C is much smaller (1-2 μm). By acting as a Ca(2+) sink, mitochondria stabilise the HCMD in space and time. In non-excitable HEK293 cells, activation of store-operated Ca(2+) entry, triggered by ER Ca(2+) emptying, also generated subplasmalemmal HCMDs, but, in this case, most of the Ca(2+) was taken up by the ER rather than by mitochondria. The smaller size of the [Ca(2+)]C peak in this case (about 2 μm) may contribute to this outcome, as the sarco-endoplasmic reticulum Ca(2+) ATPase has much higher Ca(2+) affinity than MCU. It is also possible that the relative positioning of organelles, channels and effectors, as well as cytoskeleton and accessory proteins plays an important role.

The central processes of primary nociceptors form synaptic connections with the second-order nociceptive neurons located in the dorsal horn of the spinal cord. These synapses gate the flow of nociceptive information from the periphery to the CNS, and plasticity at these synapses contributes to centrally mediated hyperalgesia and allodynia. Although exocytosis and synaptic plasticity are controlled by Ca(2+) at the release sites, the mechanisms underlying presynaptic Ca(2+) signalling at the nociceptive synapses are not well characterized. We examined the presynaptic mechanisms regulating Ca(2+) clearance following electrical stimulation in capsaicin-sensitive nociceptors using a dorsal root ganglion (DRG)/spinal cord neuron co-culture system. Cytosolic Ca(2+) concentration ([Ca(2+)]i) recovery following electrical stimulation was well approximated by a monoexponential function with a ∼2 s. Inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPase did not affect presynaptic [Ca(2+)]i recovery, and blocking plasmalemmal Na(+)/Ca(2+) exchange produced only a small reduction in the rate of [Ca(2+)]i recovery (∼12%) that was independent of intracellular K(+). However, [Ca(2+)]i recovery in presynaptic boutons strongly depended on the plasma membrane Ca(2+)-ATPase (PMCA) and mitochondria that accounted for ∼47 and 40%, respectively, of presynaptic Ca(2+) clearance. Measurements using a mitochondria-targeted Ca(2+) indicator, mtPericam, demonstrated that presynaptic mitochondria accumulated Ca(2+) in response to electrical stimulation. Quantitative analysis revealed that the mitochondrial Ca(2+) uptake is highly sensitive to presynaptic [Ca(2+)]i elevations, and occurs at [Ca(2+)]i levels as low as ∼200-300 nm. Using RT-PCR, we detected expression of several putative mitochondrial Ca(2+) transporters in DRG, such as MCU, Letm1 and NCLX. Collectively, this work identifies PMCA and mitochondria as the major regulators of presynaptic Ca(2+) signalling at the first sensory synapse, and underlines the high sensitivity of the mitochondrial Ca(2+) uniporter in neurons to cytosolic Ca(2+).

Mitochondria are key organelles involved in many aspects of plant physiology and, their ability to generate specific Ca²⁺ signatures in response to abiotic and biotic stimuli has been reported as one of their roles. The recent identification of the mammalian mitochondrial Ca²⁺ uniporter opens a new research area in plant biology. To study the mitochondrial Ca²⁺ handling, it is essential to have a reliable probe. Here we have reported the generation of an Arabidopsis transgenic line expressing the genetically encoded probe Cameleon D3cpv targeted to mitochondria, and compared its properties with the already known Cameleon YC3.6.

Aim To study the role of mitochondria in the recovery of guinea-pig hearts exposed to high-K+-cardioplegia (CPG) and ischaemia/reperfusion (I/R) Methods We measured contractility and heat release in perfused guinea-pig hearts and cytosolic and mitochondrial Ca2+ by epifluorescence and confocal microscopy in isolated cardiomyocytes loaded with Fluo-4 or Rhod-2. Results In hearts, CPG increased the postischaemic contractile recovery, and this was potentiated by the mNCX blocker clonazepam and the mKATP opener diazoxide, which also prevented the fall in muscle economy. Moreover, CPG prevented the stunning induced by ouabain, which was reduced by clonazepam. In cardiomyocytes, CPG increased fluorescent signals of cytosolic and mitochondrial Ca2+, while the addition of a mNCX blocker (CGP37157) increased cytosolic but reduced mitochondrial [Ca2+]. Ouabain in CPG increased cytosolic Ca2+ and resting heat, but the addition of CGP37157 reduced them, as well as mitochondrial Ca2+. Conclusions CPG, diazoxide and clonazepam improve postischaemic recovery, respectively, by increasing the Ca2+ cycling and by reducing the mitochondrial Ca2+ uptake either by uniporter or by mNCX. The mitochondria compete with the leaky sarcoplasmic reticulum (SR) as sink of Ca2+ in guinea-pig hearts, affecting the postischaemic contractility. CPG also prevented the ouabain-induced dysfunction by avoiding the Ca2+ overload. Ouabain reduced the synergism between CPG and clonazepam suggesting that [Na+]i and SR load influence the mNCX role.

The uniporter TAT1 (Slc16a10) mediates the facilitated diffusion of aromatic amino acids (AAAs) across basolateral membranes of kidney, small intestine and liver epithelial cells, and across the plasma membrane of non-epithelial cells like skeletal myocytes. Its role for body AA homeostasis has now been investigated using newly generated TAT1 (Slc16a10) defective mice (tat1−/−). These mice grow and reproduce normally, show no gross phenotype and no obvious neurological defect. Histological analysis did not reveal abnormalities and there is no compensatory change in any tested AA transporter mRNA. TAT1 null mice, however, display increased plasma, muscle and kidney AAA concentration under both normal and high protein diet, although this concentration remains normal in the liver. A major aromatic aminoaciduria and a smaller urinary loss of all substrates additionally transported by l-type AA antiporter Lat2–4F2hc (Slc7a8) were revealed under a high protein diet. This suggests an epithelial transport defect as also shown by the accumulation of intravenously injected 123I-2-I-l-Phe in kidney and l-[3H]Phe in ex vivo everted gut sac enterocytes. Taken together, these data indicate that the uniporter TAT1 is required to equilibrate the concentration of AAAs across specific membranes. For instance, it enables hepatocytes to function as a sink that controls the extracellular AAAs concentration. Additionally, it facilitates the release of AAAs across the basolateral membrane of small intestine and proximal kidney tubule epithelial cells, thereby allowing the efflux of other neutral AAs presumably via Lat2–4F2hc.

Neonatal rat ventricular cardiomyocytes were used to investigate mechanisms underlying transient changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by pulsed infrared radiation (IR, 1862 nm). Fluorescence confocal microscopy revealed IR-evoked [Ca2+]i events with each IR pulse (3-4 ms pulse⁻¹, 9.1-11.6 J cm⁻² pulse⁻¹). IR-evoked [Ca2+]i events were distinct from the relatively large spontaneous [Ca2+]i transients, with IR-evoked events exhibiting smaller amplitudes (0.88 ΔF/F0 vs. 1.99 ΔF/F0) and shorter time constants (τ =0.64 s vs. 1.19 s, respectively). Both IR-evoked [Ca2+]i events and spontaneous [Ca2+]i transients could be entrained by the IR pulse (0.2-1 pulse s⁻¹), provided the IR dose was sufficient and the radiation was applied directly to the cell. Examination of IR-evoked events during peak spontaneous [Ca2+]i periods revealed a rapid drop in [Ca2+]i, often restoring the baseline [Ca2+]i concentration, followed by a transient increase in [Ca2+]i.Cardiomyocytes were challenged with pharmacological agents to examine potential contributors to the IR-evoked [Ca2+]i events. Three compounds proved to be the most potent, reversible inhibitors: (1) CGP-37157 (20 μM, n =12), an inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX), (2) Ruthenium Red (40 μM, n =13), an inhibitor of the mitochondrial Ca2+ uniporter (mCU), and (3) 2-aminoethoxydiphenylborane (10 μM, n =6), an IP3 channel antagonist. Ryanodine blocked the spontaneous [Ca2+]i transients but did not alter the IR-evoked events in the same cells. This pharmacological array implicates mitochondria as the major intracellular store of Ca2+ involved in IR-evoked responses reported here. Results support the hypothesis that 1862 nm pulsed IR modulates mitochondrial Ca2+ transport primarily through actions on mCU and mNCX.

Excess administration of glutamate is known to induce Ca(2+) overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg(2+) in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg(2+) in the excitotoxicity, we simultaneously measured intracellular Ca(2+) and Mg(2+), using fluorescent dyes, Fura red, a fluorescent Ca(2+) probe, and KMG-104, a highly selective fluorescent Mg(2+) probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg(2+) concentration ([Mg(2+)](i)). Extracellular Mg(2+) was not required for this glutamate-induced increase in [Mg(2+)](i), and no increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) or [Mg(2+)](i) was observed in neurons in nominally Ca(2+)-free medium. Application of 5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca(2+)](i) and [Mg(2+)](i). Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg(2+)](i) but did induce an additive increase in [Ca(2+)](i). Moreover, the glutamate-induced increase in [Mg(2+)](i) was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg(2+) efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca(2+) uniporter, prevented this [Mg(2+)](i) increase. These results indicate that glutamate-induced increases in [Mg(2+)](i) result from the Mg(2+) release from mitochondria and that Ca(2+) accumulation in the mitochondria is required for this Mg(2+) release.

Mitochondrial Ca(2+) handling plays an important role in energy production and various cellular signaling processes. Mitochondrial Ca(2+) uptake is regulated by the mitochondrial Ca(2+) uniporter (MCU), at least one non-MCU Ca(2+) channel and possibly a mitochondrial ryanodine receptor. Two distinct mechanisms mediate Ca(2+) outward transport, the Na(+)-dependent (mNCX) and the Na(+)-independent Ca(2+) efflux. In recent years we gained more insight into the regulation and function of these different Ca(2+) transport mechanisms. However, the precise physiological role and the molecular structure of all mitochondrial Ca(2+) transporters and channels still has to be determined.

Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na+-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.—Takanaga, H., Frommer, W. B. Facilitative plasma membrane transporters function during ER transit.

Cytosolic Ca(2+) concentration ([Ca(2+)](i)) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage-gated Ca(2+) channels restores cytosolic Ca(2+) levels and reduces this neuronal death (Snider et al. 2002). We now show that this reduction in [Ca(2+)](i) is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca(2+) influx such as activation of voltage-gated Ca(2+) channels or stimulation of Ca(2+) entry via the plasma membrane Na-Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage-gated Ca(2+) channels and a less robust increase in [Ca(2+)](i) after depolarization. Levels of endoplasmic reticulum Ca(2+) were reduced and capacitative Ca(2+) entry was impaired early in the cell death process. Mitochondrial Ca(2+) was slightly increased. Preventing the transfer of Ca(2+) from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca(2+) uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca(2+,) possibly mediated by dysfunction of voltage-gated Ca(2+) channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.

Massive amounts of Ca(2+) can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mitochondrial uniporter is the foremost pathway for Ca(2+) efflux, the release of sequestered Ca(2+) by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca(2+)-phosphate complex. In the present study, we compared the effect of stepwise depolarization on Ca(2+) release induced by either the complex III inhibitor stigmatellin or an uncoupler in energized Ca(2+)-loaded rat liver mitochondria in the presence of phosphate, at extramitochondrial pH (pH(o)) 6.8 and pH(o) 7.8. Both poisons were examined in the presence and absence of oligomycin. Prior to Ca(2+) loading, mitochondria were allowed to phosphorylate 0.5 mm ADP. Opening of the permeability transition pore was additionally hampered by cyclosporin A, and was monitored by changes in light scattering. Na(+) was excluded from the medium, preventing Na(+)/Ca(2+) exchange. At both pH(o) values, Delta pH was in the range 0.11-0.15. Complete depolarization by uncoupling with or without oligomycin resulted in an approximately pH 0.05 acidic shift, but there was none in the case of stigmatellin plus oligomycin. At pH(o) 6.8 and in the presence of oligomycin, the uncoupler-induced Ca(2+) release started in the -80 to -50 mV range, whereas in the absence of oligomycin, the release occurred at approximately -15 mV. Stigmatellin induced minor Ca(2+) release only in the presence of oligomycin, starting at approximately -4 mV. At pH(o) 7.8, the uncoupler-induced Ca(2+) release started at approximately -11 mV, irrespective of the presence or absence of oligomycin. Unexpectedly, at this alkaline pH and in the presence of oligomycin, stigmatellin induced substantial Ca(2+) release, starting at approximately -10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler-induced Ca(2+) release from mitochondria.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca2+ waves. The purpose of this study was to investigate if this activity was influenced by Ca2+ uptake into mitochondria. Spontaneous Ca2+ waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 μm) and antimycin A (5 μm) abolished Ca2+ waves and increased basal Ca2+ levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 μm) and CCCP (1 μm). Spontaneous Ca2+ waves were not inhibited by the ATP synthase inhibitor oligomycin (1 μm), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at −60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 μm) also inhibited STICS. Stimulation of Ca2+ uptake into mitochondria using the plant flavonoid kaempferol (10 μm) induced a series of propagating Ca2+ waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca2+, but was not significantly affected by the IP3 receptor blocker 2-APB (100 μm). These data suggest that spontaneous Ca2+ waves in urethral ICC are regulated by buffering of cytoplasmic Ca2+ by mitochondria.

Calcium is a key ion and is known to mediate signalling pathways between cytosol and mitochondria and modulate mitochondrial energy metabolism. To gain a quantitative, biophysical understanding of mitochondrial Ca2+ regulation, we developed a thermodynamically balanced model of mitochondrial Ca2+ handling and bioenergetics by integrating kinetic models of mitochondrial Ca2+ uniporter (CU), Na+–Ca2+ exchanger (NCE), and Na+–H+ exchanger (NHE) into an existing computational model of mitochondrial oxidative phosphorylation. Kinetic flux expressions for the CU, NCE and NHE were developed and individually parameterized based on independent data sets on flux rates measured in purified mitochondria. While available data support a wide range of possible values for the overall activity of the CU in cardiac and liver mitochondria, even at the highest estimated values, the Ca2+ current through the CU does not have a significant effect on mitochondrial membrane potential. This integrated model was then used to analyse additional data on the dynamics and steady-states of mitochondrial Ca2+ governed by mitochondrial CU and NCE. Our analysis of the data on the time course of matrix free [Ca2+] in respiring mitochondria purified from rabbit heart with addition of different levels of Na+ to the external buffer medium (with the CU blocked) with two separate models – one with a 2: 1 stoichiometry and the other with a 3: 1 stoichiometry for the NCE – supports the hypothesis that the NCE is electrogenic with a stoichiometry of 3: 1. This hypothesis was further tested by simulating an additional independent data set on the steady-state variations of matrix free [Ca2+] with respect to the variations in external free [Ca2+] in purified respiring mitochondria from rat heart to show that only the 3: 1 stoichiometry model predictions are consistent with the data. Based on these analyses, it is concluded that the mitochondrial NCE is electrogenic with a stoichiometry of 3: 1.

The heart adapts the rate of mitochondrial ATP production to energy demand without noticeable changes in the concentration of ATP, ADP and Pi, even for large transitions between different workloads. We suggest that the changes in demand modulate the cytosolic Ca2+ concentration that changes mitochondrial Ca2+ to regulate ATP production. Thus, the rate of ATP production by the mitochondria is coupled to the rate of ATP consumption by the sarcomere cross-bridges (XBs). An integrated model was developed to couple cardiac metabolism and mitochondrial ATP production with the regulation of Ca2+ transient and ATP consumption by the sarcomere. The model includes two interrelated systems that run simultaneously utilizing two different integration steps: (1) The faster system describes the control of excitation contraction coupling with fast cytosolic Ca2+ transients, twitch mechanical contractions, and associated fluctuations in the mitochondrial Ca2+. (2) A slower system simulates the metabolic system, which consists of three different compartments: blood, cytosol, and mitochondria. The basic elements of the model are dynamic mass balances in the different compartments. Cytosolic Ca2+ handling is determined by four organelles: sarcolemmal Ca2+ influx and efflux; sarcoplasmic reticulum (SR) Ca2+ release and sequestration (SR); binding and dissociation from sarcomeric regulatory troponin complexes; and mitochondrial Ca2+ flows. Mitochondrial Ca2+ flows are determined by the Ca2+ uniporter and the mitochondrial Na+Ca2+ exchanger. The cytosolic Ca2+ determines the rate of ATP consumption by the sarcomere. Ca2+ binding to troponin regulates the rate of XBs recruitment and force development. The mitochondrial Ca2+ concentration determines the pyruvate dehydrogenase activity and the rate of ATP production by the F(1)-F(0) ATPase. The workload modulates the cytosolic Ca2+ concentration through feedback loops. The preload and afterload affect the number of strong XBs. The number of strong XBs determines the affinity of troponin for Ca2+, which alters the cytosolic Ca2+ transient. Model simulations quantify the role of Ca2+ in simultaneously controlling the power of contraction and the rate of ATP production. It explains the established empirical observation that significant changes in the metabolic fluxes can occur without significant changes in the key nucleotide (ATP and ADP) concentrations. Quantitative investigations of the mechanisms underlying the cardiac control of biochemical to mechanical energy conversion may lead to novel therapeutic modalities for the ischemic and failing myocardium.

Marked accumulation of arachidonic acid (AA) and intracellular Ca2+ and Na+ overloads are seen during brain ischemia. In this study, we show that, in neurons, AA induces cytosolic Na+ ([Na+](cyt)) and Ca2+ ([Ca2+](cyt)) overload via a non-selective cation conductance (NSCC), resulting in mitochondrial [Na+](m) and [Ca2+](m) overload. Another two types of free fatty acids, including oleic acid and eicosapentaenoic acid, induced a smaller increase in the [Ca2+](i) and [Na+](i). RU360, a selective inhibitor of the mitochondrial Ca2+ uniporter, inhibited the AA-induced [Ca2+](m) and [Na+](m) overload, but not the [Ca2+](cyt) and [Na+](cyt) overload. The [Na+](m) overload was also markedly inhibited by either Ca2+-free medium or CGP3715, a selective inhibitor of the mitochondrial Na+(cyt)-Ca2+(m) exchanger. Moreover, RU360, Ca2+-free medium, Na+-free medium, or cyclosporin A (CsA) largely prevented AA-induced opening of the mitochondrial permeability transition pore, cytochrome c release, and caspase 3-dependent neuronal apoptosis. Importantly, Na+-ionophore/Ca2+-free medium, which induced [Na+](m) overload, but not [Ca2+](m) overload, also caused cyclosporin A-sensitive mitochondrial permeability transition pore opening, resulting in caspase 3-dependent apoptosis, indicating that [Na+](m) overload per se induced apoptosis. Our results therefore suggest that AA-induced [Na+](m) overload, acting via activation of the NSCC, is an important upstream signal in the mitochondrial-mediated apoptotic pathway. The NSCC may therefore act as a potential neuronal death pore which is activated by AA accumulation under pathological conditions.

Cardiac myocytes, in the intact heart, are exposed to shear/fluid forces during each cardiac cycle. Here we describe a novel Ca2+ signalling pathway, generated by ‘pressurized flows’ (PFs) of solutions, resulting in the activation of slowly developing (∼300 ms) Ca2+ transients lasting ∼1700 ms at room temperature. Though subsequent PFs (applied some 10–30 s later) produced much smaller or undetectable responses, such transients could be reactivated following caffeine- or KCl-induced Ca2+ releases, suggesting that a small, but replenishable, Ca2+ pool serves as the source for their activation. PF-triggered Ca2+ transients could be activated in Ca2+-free solutions or in solutions that block voltage-gated Ca2+ channels, stretch-activated channels (SACs), or the Na+–Ca2+ exchanger (NCX), using Cd2+, Gd3+, or Ni2+, respectively. PF-triggered Ca2+ transients were significantly smaller in quiescent than in electrically paced myocytes. Paced Ca2+ transients activated at the peak of PF-triggered Ca2+ transients were not significantly smaller than those produced normally, suggesting functionally separate Ca2+ pools for paced and PF-triggered transients. Suppression of nitric oxide (NO) or IP3 signalling pathways did not alter the PF-triggered Ca2+ transients. On the other hand, mitochondrial metabolic uncoupler FCCP, in the presence of oligomycin (to prevent ATP depletion), reversibly suppressed PF-triggered Ca2+ transients, as did the mitochondrial Ca2+ uniporter (mCU) blocker, Ru360. Reducing agent DTT and reactive oxygen species (ROS) scavenger tempol, as well as mitochondrial NCX (mNCX) blocker CGP-37157, inhibited PF-triggered Ca2+ transients. In rhod-2 AM-loaded and permeabilized cells, confocal imaging of mitochondrial Ca2+ showed a transient increase in Ca2+ on caffeine exposure and a decrease in mitochondrial Ca2+ on application of PF pulses of solution. These signals were strongly suppressed by either Na+-free or CGP-37157-containing solutions, implicating mNCX in mediating the Ca2+ release process. We conclude that subjecting rat cardiac myocytes to pressurized flow pulses of solutions triggers the release of Ca2+ from a store that appears to access mitochondrial Ca2+.

Hypoxic mammalian neurons undergo excitotoxic cell death, whereas painted turtle neurons survive prolonged anoxia without apparent injury. Anoxic survival is possibly mediated by a decrease in N-methyl-d-aspartate receptor (NMDAR) activity and maintenance of cellular calcium concentrations ([Ca(2+)](c)) within a narrow range during anoxia. In mammalian ischaemic models, activation of mitochondrial ATP-sensitive K(+) (mK(ATP)) channels partially uncouples mitochondria resulting in a moderate increase in [Ca(2+)](c) and neuroprotection. The aim of this study was to determine the role of mK(ATP) channels in anoxic turtle NMDAR regulation and if mitochondrial uncoupling and [Ca(2+)](c) changes underlie this regulation. In isolated mitochondria, the K(ATP) channel activators diazoxide and levcromakalim increased mitochondrial respiration and decreased ATP production rates, indicating mitochondria were 'mildly' uncoupled by 10-20%. These changes were blocked by the mK(ATP) antagonist 5-hydroxydecanoic acid (5HD). During anoxia, [Ca(2+)](c) increased 9.3 +/- 0.3% and NMDAR currents decreased 48.9 +/- 4.1%. These changes were abolished by K(ATP) channel blockade with 5HD or glibenclamide, Ca(2+)(c) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by activation of the mitochondrial Ca(2+) uniporter with spermine. Similar to anoxia, diazoxide or levcromakalim increased [Ca(2+)](c) 8.9 +/- 0.7% and 3.8 +/- 0.3%, while decreasing normoxic whole-cell NMDAR currents by 41.1 +/- 6.7% and 55.4 +/- 10.2%, respectively. These changes were also blocked by 5HD or glibenclamide, BAPTA, or spermine. Blockade of mitochondrial Ca(2+)-uptake decreased normoxic NMDAR currents 47.0 +/- 3.1% and this change was blocked by BAPTA but not by 5HD. Taken together, these data suggest mK(ATP) channel activation in the anoxic turtle cortex uncouples mitochondria and reduces mitochondrial Ca(2+) uptake via the uniporter, subsequently increasing [Ca(2+)](c) and decreasing NMDAR activity.

Mitochondrial Ca2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca2+ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A2 or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca2+ from extracellular medium and blockage of mitochondrial Ca2+ uptake by either a mitochondrial uncoupler or a Ca2+ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca2+ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca2+-dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca2+ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.

Background and purpose: The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes. Experimental approach. [Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells. Key results: KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concentrations required to inhibit MCU. Conclusions and Implications: Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future. British Journal of Pharmacology (2007) 151, 647–654; doi:10.1038/sj.bjp.0707260

The recent availability of activators of the mitochondrial Ca(2+) uniporter allows direct testing of the influence of mitochondrial Ca(2+) uptake on the overall Ca(2+) homeostasis of the cell. We show here that activation of mitochondrial Ca(2+) uptake by 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca(2+) release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na(+)-Ca(2+) exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca(2+) uptake and release from mitochondria control the ability of local Ca(2+) microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). In addition, the ability of mitochondria to control Ca(2+) release from the ER allows them to modulate cytosolic Ca(2+) oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca(2+) oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca(2+) oscillations are exquisitely sensitive to the rates of mitochondrial Ca(2+) uptake and release, which precisely control the size of the local Ca(2+) microdomains around InsP(3)Rs and thus the ability to produce feedback activation or inhibition of Ca(2+) release.

Before dilution in hypoosmotic media sperm of freshwater fish are maintained quiescent by a range of factors including osmolality, K+ and pH, and the onset of motility is generally associated with an increase in cytoplasmic Ca2+. In contrast, Ca2+ in conjunction with osmolality was found to inhibit motility of intact bluegill sperm. Consistent with seminal plasma composition, 0.16 mmol/L Ca2+ and greater, in conjunction with an osmotic concentration of 290 mOsm/kg, inhibited the onset of bluegill sperm motility; sperm diluted in saline at 290 mOsm/kg without Ca2+ became motile. Cations Mn2+ and Sr2+, in conjunction with osmolality, had an inhibitory effect on initiation of sperm motility similar to that of Ca2+. Sperm motility was inhibited by Ca2+ channel blockers nimodipine and nifedipine, the mitochondrial Ca2+ uniporter inhibitor ruthenium red and the calmodulin inhibitors W-7 and trifluoperazine dihydrochloride. These results provide evidence that elevated cytoplasmic Ca2+ inhibits sperm motility and yet low levels permit or promote motility. This study demonstrates a unique inhibitory action of Ca2+ on the motility of intact fish sperm at physiologically relevant levels. J. Exp. Zool. 307A:590–599, 2007. © 2007 Wiley-Liss, Inc.

Our previous work has demonstrated that while the Ca(2+) and Pi ions acting in concert function as a potent osteoblast apoptogen, the underlying mechanisms by which it activates cell death is not known. We hypothesize that the ion pair causes release of Ca(2+) from intracellular stores ([Ca(2+)]i); the increase in intracellular calcium prompts the mitochondria to uptake more calcium. This accumulation of calcium eventually results in the loss of mitochondrial membrane potential (MMP) and, subsequently, apoptosis. To test this hypothesis, we evaluated apoptosome formation in MC3T3-E1 osteoblast-like cells treated with the ion pair. Western blot analysis indicated migration of cytochrome-c and Smac/DIABLO from mitochondria to the cytoplasm. Inhibition of either the electron transfer chain (with antimycin a and rotenone), or the activation of a MMP transition (with bongkrekic acid) inhibited apoptosis in a dose-dependent manner. Pre-treating osteoblasts with ruthenium red, a Ca(2+) uniporter inhibitor of both mitochondria and the endoplasmic reticulum (ER), also completely abolished Ca(2+.)Pi-induced apoptosis. Moreover, we showed that an increase in [Ca(2+)]i preceded the increase in MMP over the first 45 min of treatment; a mitochondrial membrane permeability transition was evident at 75 min. To determine the role of ER, Ca(2+) stores in the generation of the apoptotic signal by the ion pair, cells were treated with several inhibitors. Apoptosis was inhibited when cells were treated with dantrolene, an inhibitor of ER ryanodine receptors, and 2-aminodiphenylborate, an IP3 Ca(2+) channel inhibitor, but not cyclopiazonic acid, an ER Ca(2)-ATPase inhibitor. Together, these data demonstrate that Ca(2+) Pi-induced osteoblast apoptosis is characterized by the generation of an apoptosome and that Ca(2+) release from ER stores may promote ion pair-dependent cell death.

The Ca2+ influx into mitochondrial matrix is primarily mediated via the mitochondrial calcium uniporter (MCU). Basal tyrosine phosphorylation (P-Y) of MCU was shown in mass spectroscopy of human ti...

Ca(2+) uptake by mitochondria is a key element in the control of cellular Ca(2+) homeostasis and Ca(2+)-dependent phenomena. It has been known for many years that this Ca(2+) uptake is mediated by the mitochondrial Ca(2+) uniporter, a specific Ca(2+) channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter. The specific alpha-ER agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca(2+) uptake in permeabilized HeLa cells by 10-fold at 2 microM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca(2+)] peak and reduced the cytosolic one. Diethylstilbestrol and 17-beta-estradiol (but not 17-alpha-estradiol) were active at pharmacological concentrations while the beta-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective. The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca(2+) uptake (IC(50) 2.5+/-1.5 and 2.5+/-1.4 microM, mean+/-s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect. Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum. Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca(2+) uptake. These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca(2+) uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.

Current evidence suggests that zinc kills neurons by disrupting energy production, specifically by inhibiting mitochondrial function. However it is unclear if the inhibitory effect requires zinc accumulation, and if so, precisely how zinc enters mitochondria. Here, using fluorescence microscopy to visualize individual rat brain mitochondria, we detected matrix zinc uptake using the fluorophore FluoZin-3. Fluorescence increased rapidly in mitochondria treated with micromolar free zinc, and was quickly returned to baseline by membrane permeant chelation. Zinc uptake occurred through the calcium uniporter, because depolarization or uniporter blockade reduced fluorescence changes. However, increased fluorescence under these conditions suggests that zinc can enter through a uniporter-independent pathway. Fluorescence steadily declined over time and was unaffected by acidification or phosphate depletion, suggesting that zinc precipitation is not a mechanism for reducing matrix zinc. Uniporter blockade with ruthenium red also did not change the rate of zinc loss. Instead, zinc appears to exit the matrix through a novel efflux pathway not yet identified. Interestingly, dye-loaded mitochondria showed no fluorescence increase after treatment with strong oxidants, arguing against oxidant-labile intra-mitochondrial zinc pools. This study is the first to directly demonstrate zinc accumulation in individual mitochondria and provides insight about mechanisms mediating mitochondrial zinc uptake and efflux.

Calcium-mediated signaling regulates nuclear gene transcription by calcium/cAMP response element binding protein (CREB) via calcium-dependent kinases and phosphatases. This study tested the hypothesis that CREB is also present in mitochondria and subject to dynamic calcium-dependent modulation of its phosphorylation state. Antibodies to CREB and phosphorylated CREB (pCREB) were used to demonstrate the presence of both forms in isolated mitochondria and mitoplasts from rat brain. When energized mitochondria were exposed to increasing concentrations of Ca2+ in the physiological range, pCREB was lost while total CREB remained constant. In the presence of Ru360, an inhibitor of the mitochondrial Ca2+ uptake uniporter, calcium-dependent loss of pCREB levels was attenuated, suggesting that intramitochondrial calcium plays an important role in pCREB dephosphorylation. pCREB dephosphorylation was not, however, inhibited by the phosphatase inhibitors okadaic acid and Tacrolimus. In the absence of Ca2+, CREB phosphorylation was elevated by the addition of ATP to the mitochondrial suspension. Exposure of mitochondria to the pore-forming molecule alamethicin that causes osmotic swelling and release of intermembrane proteins enriched mitochondrial pCREB immunoreactivity. These results further suggest that mitochondrial CREB is located in the matrix or inner membrane and that a kinase and a calcium-dependent phosphatase regulate its phosphorylation state.

Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP. By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM. Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid. CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190. Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis. British Journal of Pharmacology (2004) 141, 263–268. doi:10.1038/sj.bjp.0705609

Summary The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspar- tate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identi- fied Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomy- ces cerevisiae . On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

When motor nerve terminals are stimulated repetitively, cytosolic [Ca2+] increases to a plateau amplitude that is maintained as long as stimulation persists (Wu & Betz, 1996; Ravin et al. 1997; David et al. 1998, David & Barrett, 2000; Suzuki et al. 2000). Mitochondrial Ca2+ uptake contributes importantly to limiting the increase in cytosolic [Ca2+], because when this uptake is inhibited by depolarizing the electrical potential across the inner mitochondrial membrane (Ψm), cytosolic [Ca2+] increases to much higher levels during stimulus trains (Tang & Zucker, 1997; David et al. 1998; David & Barrett, 2000; Suzuki et al. 2002). Similar effects of Ψm depolarization on stimulation-induced cytosolic [Ca2+] responses have also been observed in other nerve terminals, neuronal somata and certain secretory cells (Friel & Tsien, 1994; Steunkel, 1994; Werth & Thayer, 1994; White & Reynolds, 1995; Herrington et al. 1996; Sidky & Baimbridge, 1997; Medler & Gleason, 2002). Mitochondrial Ca2+ uptake is passive, mediated by a uniporter in the inner mitochondrial membrane and powered mainly by the Ψm created by the electron transport chain (reviewed in Gunter & Pfeiffer, 1990). This study investigated how mitochondrial Ca2+ uptake affects quantal transmitter release from mouse motor nerve terminals, using Ψm depolarizing drugs to block this uptake. Previous studies reported that these drugs increase the rate of asynchronous release recorded from resting frog, snake, crayfish and cockroach motor terminals, and increase the quantal content of phasic release evoked by nerve stimulation (Alnaes & Rahamimoff, 1975; Washio, 1982; Molgo & Pecot-Dechavassine, 1988; Zengel et al. 1994; Tang & Zucker, 1997; Calupca et al. 2001). Both of these effects were attributed to an increase in cytosolic [Ca2+], which would be predicted to increase asynchronous release directly, and to increase phasic release by a ‘residual Ca2+’ mechanism such as that postulated to mediate short-term historical processes such as facilitation. However, the studies of phasic release in vertebrate nerve terminals reduced Ca2+ influx into stimulated terminals by using low bath [Ca2+] to prevent muscle contraction. In addition, none of these preceding studies measured asynchronous release during the stimulus train, when the large increase in cytosolic [Ca2+] during Ψm depolarization might be predicted to have its major effect. We present evidence that blocking mitochondrial Ca2+ uptake by Ψm depolarization markedly increases both EPP depression and asynchronous release during 50 Hz stimulation. Therefore, the ability of mitochondria to sequester Ca2+ and thus prevent large increases in cytosolic [Ca2+] is essential to prevent desynchronization of release during repetitive stimulation.

The mechanisms of Ca2+-induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl-based media 1.4 micromol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+-induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 microM glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+ in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.

Manganese (Mn) is a neurotoxin causing Manganism in people exposed to high environmental levels. Oxidative stress is implicated as a factor of Mn toxicity. The mechanisms is attributed to toxic levels of free radicals inducing mitochondrial dysfunction. Reports indicate Mn accumulates in mitochondria, however controversy exists to the extent of Mn accumulation in mitochondria and whether this contributes to the cause of Manganism. We have been using the oyster, Crassostrea virginica, to study Mn neurotoxicty. We found Mn disrupts the animals’ dopaminergic system and mitochondrial respiration. Mn is believed to use the mitochondrial Ca2+ uniporter and H+ or Na+/Ca2+ exchanger for movements in and out of mitochondria. We hypothesize Ca2+ channel blockers will alter mitochondrial Mn accumulations. We prepared mitochondrial fractions from gills. Mitochondria were treated with Ca2+ channel blockers prior to Mn exposure, pelleted and washed, then digested in HNO3 and analyzed in an AA. Mitochondria treated with...